It is now important to distinguish between adenocarcinoma and squamous cell carcinoma of the lung because of target-specific treatments. Our study aimed to study the efficiency of Thyroid Transcription Factor-1 (TTF-1), cytokeratin 5/6 (CK5/6) and p63 in distinguishing between adenocarcinoma and squamous cell carcinoma and to study the contribution of these markers to the diagnosis in non-small cell lung cancer.Immunohistochemically, TTF-1, CK 5/6 and p63 were used in 72 cases of squamous cell carcinoma, 19 cases of adenocarcinoma, and 29 cases of non-small cell lung cancer whose final diagnosis was decided with the subsequent resection material. The specificity, sensitivity, and positive and negative predictive value were calculated for each marker.TTF-1 positivity was seen in none of the 72 squamous cell carcinomas but in all of 19 adenocarcinoma cases. CK5/6 negativity was seen in all cases of adenocarcinoma and in two cases of squamous cell carcinoma. p63 was positive in all squamous cell carcinomas and in 4 adenocarcinomas. Cytokeratin 5/6, p63 positivity and TTF-1 negativity were observed in 17 non-small cell lung cancers whose final diagnosis was squamous cell carcinoma. None of the 12 non-small cell lung cancers whose final diagnosis was adenocarcinoma exhibited positive staining for CK5/6. However, p63 staining was not seen in the biopsy but was focal in the surgical specimen in one case. All the 12 non-small cell lung cancers whose certain diagnosis was adenocarcinoma were positive for TTF-1. TTF-1, CK 5/6 and p63 seem to be useful for differentiating adenocarcinoma from squamous cell carcinoma with 100% specificity, 100% sensitivity and 100% specificity, 97% sensitivity and 87% specificity, and 100% sensitivity, respectively.We concluded that TTF-1 is a reliable marker for subtyping lung cancer. Different staining patterns can be seen with CK5/6 and p63; however, if they are used together with TTF-1 and interpreted correctly, they can be of help for the final diagnosis even in cases in which the morphology is unclear.